Assisted reproductive techniques (ART) such as in vitro fertilization (IVF) often require the preservation of oocytes and zygotes. Currently, approximately 1.5 million assisted reproductive procedures are performed worldwide. According to the American Society of Reproductive Medicine, IVF procedures cost a patient over $12,000 on average. Thus, increasing the number and quality of preserved oocytes and embryos would improve the success of assisted reproductive techniques such as IVF and reduce patient costs. In addition, it has been recently estimated that 1 in every 47 women will develop some form of invasive cancer within their reproductive age. As efforts in the scientific community continue to improve cancer survival, there is an ever-growing demand to preserve oncofertility for young women of reproductive age. The approaches for addressing both of these issues involve cryopreservation of oocytes or zygotes, which typically involves one of two conventional methods: slow-rate freezing or vitrification. In recent years, vitrification has provided increased cryosurvival of oocytes and zygotes as measured by a lower percentage of cell death post-warming and better time efficiency than other methods.
Vitrification is a process of freezing used to preserve oocytes (e.g., eggs) and embryos used for assisted reproductive therapies (see, e.g., Rall & Fahy (1985) Nature 313: 573-75). During conventional methods of manual vitrification, the oocytes and/or embryos are exposed to solutions with increasing concentrations of cryopreservative agents that prevent the formation of ice crystals (id). However, the viability of the oocytes and embryos is reduced as a result of the osmotic shock produced by the sudden volume change due to loss of water caused by these agents (Prentice-Biensch et al (2012) Reproductive Biology and Endocrinology 10:73).
Due to the thermodynamics of the vitrification process, vitrification solutions comprising high concentrations of cryoprotectant agents (CPA) are typically used to avoid ice formation during the non-equilibrium phase change from liquid to glass phase. As a result of their large size and low surface-to-volume ratio, transfer of oocytes and zygotes from culture media (CM) conditions at physiologic osmolality to a vitrification solution (VS) with high osmolality produces deleterious osmotic stress on the oocytes and zygotes. In addition, while the increase in osmolality produced by cryoprotectant agents is thermodynamically favorable for vitrification, the increase in osmolality also decreases cell volume from loss of cell liquid.
Early studies on osmotic stress indicated that at some limiting minimum cell volume, the osmotic stress becomes sufficient to cause lethal damage to cell membrane integrity. This concept of a limiting minimum cell volume has formed the basis of numerous osmotic stress studies. As a result, the conventional standard method for oocyte and zygote vitrification involves a 3-step equilibration process in which the oocytes or zygotes are manually pipetted into three subsequently higher levels of CPA concentrations. This procedure avoids sudden changes of osmolality by spreading the osmotic stress over an extended period of time and avoids the critical minimum cell volume by allowing the cell to shrink slowly and sometimes to re-expand. Further studies have shown that increasing the number of equilibration steps increases cryosurvival. However, protocols with large numbers of equilibration steps have not been clinically adopted due to the impracticality of performing many manual pipetting steps.
Accordingly, cryoprotection of biological materials such as oocytes and embryos would benefit from improved methods that decrease the osmotic shock during vitrification to increase cell health and developmental competence without increasing user involvement and accompanying user error in the process.